Epstein-Barr virus (EBV) causes infectious mononucleosis and is causally associated with two kinds of B-cell lymphomas and carcinoma in humans. In cell culture EBV infects and immortalizes human B-lymphocytes efficiently. It is likely that EBV's pathogenicity at least for its associated lymphoid diseases results from its capacity to immortalize B-cells. In this grant application we propose to refine and extend our genetic analysis of EBV's immortalizing functions. We have developed a method to study EBV genetically (1) that uses EBV-derived vectors that contain all the viral cis-acting signals required for them to participate in the lytic phase of the viral life cycle. Introduction of these vectors into appropriate cells followed by induction of the lytic cycle of an endogenous EBV allows the vectors to be amplified and packaged. These packaged vectors can infect human B-lymphocytes (1). This scheme will be optimized to yield maximum levels of the packaged vectors or maximum levels of recombinants between the vectors and the endogenous, helper EBV. The optimized scheme will be used to identify viral genes in addition to that encoding the EBV nuclear antigen-2 (EBNA-2) that are required for immortalization. Three extensions of this genetic scheme will be used to make these identifications. In addition, two approaches will be pursued to make helper cells - cells that provide helper viral functions but fail to pack-age their endogenous viral DNA. Helper cells would greatly facilitate a genetic analysis of EBV. Finally we propose to use our scheme to analyze the EBNA-2 gene which has been shown to be required for the immortalization of human B-lymphocytes by EBV (1).